Binding of Epo to the EpoR is crucial for the production of circulating erythrocytes. Our studies will critically test a hypothesis, supported by much data from my laboratory, that the three major signaling pathways induced by the EpoR- Ras/MAP kinase, STAT5, PI-3' kinase/Akt, and perhaps others-function in different ways but additively and synergistically to prevent apoptosis of erythroid progenitors. This allows them to express a terminal program of proliferation and erythroid differentiation. The loss of any pathway through mutation should reduce but not abolish survival of erythroid progenitors, leading to increased apoptosis of progenitors, fetal anemia, and the inability of the adult to respond to anemia. Indeed we showed that STAT5a-/-b/- mice exhibit all of these properties and that STAT5a-/--b/- mice exhibit all of these properties and that STAT5 induces expression of the antiapoptotic protein bclx. We propose to determine whether and how the Ras/MAP kinase and PI-3' kinase/Akt pathways similarly prevent apoptosis of cultured fetal liver erythroid progenitors or have some other function. We will determine the roles of two Akt targets, Forkhead and GATA-1, in EpoR antiapoptotic signaling. Our main focus is to determine whether each of these three Epo-induced signaling pathways prevent apoptosis of erythroid progenitors in vivo or subsume another function. We will first assemble and study in cultured cells a set of mutant EpoRs that activate none, one, or two of these pathways. Now, assemble and study in cultured cells a set of mutant EpoRs that activate none, one, or two of these pathways. Now we are generating a "knock-in" mice in which the wild- type EpoR is replaced by a mutant that activates only the PI-3' kinase/Akt pathway, 9r all pathways except the PI-3' kinase/Akt pathway. Later we shall generate mice expressing mutant EpoRs that activate only the STAT5 or the Ras MAP/kinase pathways or two of the three pathways. Overall development of the mice and the level and structure of red cells will be measured, as well as the pathways. Overall development of the mice and the level and structure of red cells will be measured, as well as the number and Epo responsiveness of erythroid progenitors in the fetus and adult. We will determine whether the erythropoietic compartment in adult mice is able to respond to the stress of anemia and whether the failure to induce a specific signaling pathway causes reduced induction of certain anti-apoptotic proteins and an increase in apoptosis of erythroid progenitors. Similar knock-in mice will confirm or refute our conclusion (derived from studies in fetal liver cells) that the cytosolic domains of the TpoR and PrlR receptors can functionally replace that of the EpoR. Finally, and in collaboration with Dr. Leonard Zon, we will begin an analysis of EpoR expression and signaling in the Zebrafish by isolating the Zebrafish by isolating the Zebrafish Epo and EpoR genes and mapping the chromosomal locations. This work will be done in part during a sabbatical by Dr. Lodish in Dr. Zon's laboratory.